Presence Of Topoisomerase Research Articles

The recombinant product of the Chryptomonas phi plastid gene hlpA is an architectural HU-like protein that promotes the assembly of complex nucleoprotein structures.

The HlpA protein which is encoded by the hlpA gene in the plastid genome of the cryptomonad alga Chryptomonas phi is structurally related to the non-sequence-specific DNA-binding and DNA-bending HU family of chromatin-associated proteins. The expression of the HlpA protein complements the mutant phenotype of Bacillus subtilis cells impaired in the Hbsu protein (B. subtilis HU), as measured by the resistance of the cells to methylmethane sulphonate. To analyse the interactions of HlpA with DNA, we expressed the protein in Escherichia coli and purified it to homogeneity. HlpA interacts preferentially with four-way junction DNA or DNA minicircles, when compared with linear DNA, recognising DNA structure. HlpA and E. coli HU display comparable affinities for all types of DNA tested; however, HlpA exhibits a stronger tendency to self-associate in the presence of DNA. Accordingly, HlpA oligomerises more readily than HU in protein crosslinking experiments. In the presence of topoisomerase I, HlpA constrains negative superhelical turns in closed circular plasmid DNA. The HlpA protein mediates the joining of distant recombination sites into a complex nucleoprotein structure, as judged by beta-mediated site-specific recombination. The results presented provide evidence that HlpA is a functional plastid equivalent of nuclear and mitochondrial HMG1-like proteins and bacterial HU proteins.

Measurement of the frequency of histone displacement during the in vitro transcription of nucleosomes: RNA is a competitor for these histones.

Transcription through tandemly arranged nucleosomes was studied to determine the frequency at which the nucleosomes would disrupt and cause displacement of the associated histones to a competitor DNA. In order to more effectively preserve topological effects, the template that was used in the in vitro transcription system was a large covalently, closed circular plasmid (8.9 kb). The plasmid contained two promoters for T7 RNA polymerase, each separated by 4.4 kb, and transcription was done in the presence of topoisomerase I at physiological ionic strength. Nucleosome disruption was observed at an approximate frequency of 1 in 4 nucleosomes such that after several rounds of transcription on the plasmid 80% of the nucleosomes were disrupted. Unexpectedly, all four histones were found associated with the RNA rather than the competitor DNA. The histones bound the competitor DNA only after removal of the RNA by RNase A treatment. By analyzing the topological state of the competitor DNA, it was observed that the majority of the histones that were displaced from the RNA were able to re-form nucleosomes. Additional experiments were done to determine the reasons for the preferential binding of histones to the newly synthesized RNA. It was found that the large molecular weight RNA binds histones with an approximate 100-fold greater affinity relative to DNA when at physiological ionic strength. Within the cell, this high-affinity binding would be expected to require cellular mechanisms to regulate the interaction of RNA with histones. The relatively high frequency of displacement of all four histones during transcription is higher than what is observed in vivo and suggests that additional factors are needed to regulate this displacement. These observations are discussed and compared with previous studies that have examined the process of transcription through nucleosomes.

The acidic tail of the high mobility group protein HMG-D modulates the structural selectivity of DNA binding

HMG-D is one of the Drosophila counterparts of the vertebrate HMG1/2 class of abundant chromosomal proteins and contains three domains: an HMG domain followed by a basic region and a short acidic carboxy-terminal tail. We show that the HMG domain of HMG-D does not bind to deformed DNA structures such as DNA bulges, cis-platinated DNA or four-way junctions but does bind tightly to DNA microcircles, suggesting that in vivo the natural ligands of this domain are tightly bent DNA loops. The flanking basic region substantially increases the DNA-binding activity of the HMG domain to DNA ligands other than microcircles. We demonstrate that the acidic tail alters the structural selectivity of DNA binding by increasing the affinity for deformed DNA and decreasing the affinity for linear B-DNA. Finally, we show that the acidic tail increases the efficiency of constraining preformed negative supercoils but conversely decreases the efficiency of supercoiling relaxed DNA in the presence of topoisomerase I.

From amsacrine to DACA (N-[2-(dimethylamino)ethyl]acridine-4-carboxamide): Selectivity for topoisomerases I and II among acridine derivatives

A number of acridine derivatives, including the clinical antileukaemia agent amsacrine and the experimental agent DACA (N-[2-(dimethylamino)ethyl]acridine-4-carboxamide), target the enzyme topoisomerase II. We demonstrate here that DACA induces DNA cleavage in the presence of topoisomerase I as well as of topoisomerase II. We also investigate a series of acridine derivatives which link amsacrine to DACA in terms of DNA binding, topoisomerase poisoning and biological activity. The presence of an acridine 4-linked N-2-(dimethylamino)ethyl group provides both a pronounced G-C preference for DNA binding and activity towards topoisomerase I. The removal of the anilino side chain of amsacrine, in combination with the presence of the N-2-(dirnethylamino)ethyl group, provides in vitro biological activity against “atypical” multidrug resistant leukaemia lines with low topoisomerase II activity. Among these compounds, suppression of the ionisation of the acridine nitrogen to produce the compound DACA is associated with experimental activity against solid tumours. The addition of an acridine 2-chloro substituent to DACA suppresses the stimulation of topoisomerase II-dependent DNA cleavage but increases stimulation of topoisomerase I cleavage. 2-Substitution also increases activity against the “atypical” multidrug resistant cell lines. Overall, the results suggest that augmentation of topoisomerase I-dependent activity in this series by appropriate chemical substitution in this series leads to circumvention of topoisomerase II-mediated multidrug resistance.

Synthesis and evaluation of terbenzimidazoles as topoisomerase I inhibitors.

The synthesis and pharmacological activity of a series of terbenzimidazoles are described. The ability of these derivatives to induce DNA cleavage in the presence of topoisomerase I was evaluated in vitro. These analogs were also assayed for their cytotoxicity in RPMI 8402 cells and the camptothecin-resistant CPT-K5 cells. In addition the potential for these compounds to serve as substrates for MDR1 was also determined. Several terbenzimidazoles exhibited similar cytotoxicity against variants of human tumor cells that either overexpress MDR1 or are camptothecin-resistant.

Preferential binding of histones H3 and H4 to highly positively coiled DNA.

The interaction of histones H3 and H4 with highly positively coiled DNA has been studied. To carry out this study, it was necessary to develop a protocol for the preparation of large quantities of highly positively coiled DNA. Such preparations were obtained by maximizing transcription-induced stress in cells containing PBR322 and then applying a short-term treatment with novobiocin, which inhibits gyrase. Fractionation on CsCl-EtBr gradients gave PBR322 plasmids with topological states ranging from +0.15 to -0.043 superhelical density. In competition experiments between negatively and positively coiled DNA, histones H3,H4 preferentially bound the positively coiled DNA when the superhelical density was greater than +0.10. This preference was shown on the basis of sedimentation rate on sucrose gradients, selective aggregation by H3,H4 binding, and cross-linking experiments in which the histone-DNA content was characterized on CsCl density gradients. An analysis of the DNA helical pitch in 1.1 M NaCl, a condition in which moderately positively coiled DNA preferentially binds H3,H4 indicated that the preferential binding may be due to a decrease in helical pitch that approximates 0.06 bp/turn. Consistent with this observation is the ability of histones H3,H4 to transiently hold this altered pitch in the presence of topoisomerase I. A possible explanation for this preference is based on the known observation that histones overwind the DNA helix when in a nucleosome. These data provide an explanation for the in vivo observation that histones H3,H4 are rarely displaced during the transcription process. These observations are discussed with regard to mechanisms for transcription through nucleosomes.

Localization of an aminoacridine antitumor agent in a type II topoisomerase-DNA complex.

Type II topoisomerases are the targets of several classes of chemotherapeutic agents that stabilize an intermediate of the catalytic cycle with the enzyme covalently linked to cleaved DNA. We have used 3-azido-AMSA [4'-(3-azido-9-acridinylamino)methanesulfon-m-anisidide], a photo-activatible analog of the inhibitor m-AMSA [4'-(9-acridinylamino)methanesulfon-m-anisidide], to localize the inhibitor binding site in a cleavage complex consisting of an oligonucleotide substrate and the bacteriophage T4 type II DNA topoisomerase. Upon photoactivation, the inhibitor covalently attached to the substrate only in the presence of topoisomerase. Sites of inhibitor attachment were detected by primer-extension analysis and by piperidine-induced cleavage of the covalently modified substrate. 3-Azido-AMSA reacted with bases immediately adjacent to the two phosphodiester bonds cleaved by the enzyme. Therefore, topoisomerase creates or stabilizes preferential binding sites for the inhibitor precisely at the two sites of DNA cleavage.

Kinetics of chromosome condensation in the presence of topoisomerases: a phantom chain model

We discuss the requirement of type II DNA topoisomerase in the process of mitotic chromosome condensation. Using a known model describing the collapse of homopolymers, we propose that the compaction process necessitates a change in the topological state (i.e., a self-knotting) of the chromosomal chain. We argue that the enzymes are necessary to reach the compact metaphase state in a time interval that is much smaller than the time expected in the uncatalyzed process. The folding process is such that the potential entanglement points are localized at particular regions of the chromosome known as the scaffold-associated regions. The concentration of entanglements in the metaphase chromosome is related to the average size of the radial loops. A phantom chain model for the condensation process, in which each potential entanglement point is dealt with by a topoisomerase II molecule, is proposed.

DNA looping by the HMG-box domains of HMG1 and modulation of DNA binding by the acidic C-terminal domain.

We have compared HMG1 with the product of tryptic removal of its acidic C-terminal domain termed HMG3, which contains two 'HMG-box' DNA-binding domains. (i) HMG3 has a higher affinity for DNA than HMG1. (ii) Both HMG1 and HMG3 supercoil circular DNA in the presence of topoisomerase I. Supercoiling by HMG3 is the same at approximately 50 mM and approximately 150 mM ionic strength, as is its affinity for DNA, whereas supercoiling by HMG1 is less at 150 mM than at 50 mM ionic strength although its affinity for DNA is unchanged, showing that the acidic C-terminal tail represses supercoiling at the higher ionic strength. (iii) Electron microscopy shows that HMG3 at a low protein:DNA input ratio (1:1 w/w; r = 1), and HMG1 at a 6-fold higher ratio, cause looping of relaxed circular DNA at 150 mM ionic strength. Oligomeric protein 'beads' are apparent at the bases of the loops and at cross-overs of DNA duplexes. (iv) HMG3 at high input ratios (r = 6), but not HMG1, causes DNA compaction without distortion of the B-form. The two HMG-box domains of HMG1 are thus capable of manipulating DNA by looping, compaction and changes in topology. The acidic C-tail down-regulates these effects by modulation of the DNA-binding properties.

Studies on the ability of minor groove binders to induce supercoiling in DNA

The effect of various non-intercalating minor groove binders on closed circular DNA in the presence of topoisomerase I has been studied by means of agarose gel electrophoresis. Analogues of the netropsin series (lexitropsins) and SN-6999 can effectively produce positive supercoils, as indicated by analysis of the topoisomers in the presence of chloroquine and the evaluated linking number changes. Analogues of the distamycin series are less effective, and bisquaternary ammonium heterocycles, as well as DAPI and pentamidine, were found to be ineffective ligands. The large differences observed in the ability of minor groove binders to induce positive supercoils are discussed.

Inhibition of topoisomerase I function by nitidine and fagaronine.

The benzophenanthridine alkaloids nitidine and fagaronine were characterized as inhibitors of topoisomerase I function. In common with the antitumor agent camptothecin, both nitidine and fagaronine stabilized the covalent binary complex formed between calf thymus topoisomerase I and DNA. The effects of these compounds were readily apparent at 0.15-0.3 microM concentrations. Both nitidine and fagaronine inhibited the topoisomerase I-mediated relaxation of supercoiled pSP64 plasmid DNA more effectively than camptothecin; unlike camptothecin, both of these benzophenanthridine alkaloids also bound directly to and mediated the unwinding of B-form DNA. Nitidine and fagaronine were also studied in comparison with camptothecin to determine the sequence specificity of DNA breaks produced from a 32P-end-labeled duplex in the presence of topoisomerase I. All three compounds produced very similar cleavage patterns. The specificity of nitidine and fagaronine for inhibiting topoisomerase I function was studied by measuring the effects of the compounds on the unknotting of P4 DNA by calf thymus topoisomerase II. Moderate inhibition of topoisomerase II-mediated unknotting was obtained, but only in the presence of high (i.e., 40 microM) concentrations of nitidine and fagaronine. In comparison, doxorubicin inhibited topoisomerase II to the same extent as nitidine and fagaronine when it was employed at 2.5 microM concentration and was strongly inhibitory when employed at 10 microM concentration.

Altered topoisomerase I activity and recombination activating gene expression in a human leukemia cell line resistant to doxorubicin

We examined the expression of the genes encoding topoisomerases I and II and those associated with V(D)J [variable(diversity)joining] recombination in two human T-cell acute lymphoblastic leukemia (T.ALL) cell lines, CEM and CEM/DOX. In CEM/JDOX cells, which are resistant to doxorubicin, the topoisomerase I gene was found to be 4-fold overexpressed and nuclear topoisomerase I relaxation activity was 2-fold greater in CEM/DOX than in CEM cells. Furthermore, the cleavable complex reaction induced by camptothecin, a specific topoisomerase I inhibitor, was found to be 2.5-increased in the presence of topoisomerase I extracted from CEM/DOX, in comparison to that in CEM cells. Conversely, the topoisomerase II mRNA levels, nuclear decatenation activities and (mAMSA) 4′(9-acridinylamino)methanesulfon- m-anisidide-induced cleavable complex formation in CEM/DOX were similar to those of the doxorubicin-sensitive cells. The results indicate that topoisomerase I activity is elevated in CEM/DOX cells. Nevertheless, CEM/DOX cells were 11-fold more resistant to camptothecin than were CEM cells, and cross-resistance to camptothecin was not reversed by verapamil. Furthermore, using an intact cell assay for DNA-protein complexes, we found that camptothecin-stimulated cleavable complexes formed in CEM/DOX cells were increased in correlation with the elevated topoisomerase I activity. These results suggest that camptothecin resistance in CEM/DOX cells is due to different mechanism(s) than topoisomerase- or P-glycoprotein-associated multidrug resistance. The recombination activating gene, RAG1, which is one of the components of the site-specific V(D)J recombination complex, was 20-fold overexpressed in CEM/DOX cells. In contrast, RAG2 and T160 gene transcripts, other components of the V(D)J complex, were at best poorly detected in both sensitive and resistant cells. No specific V(D)J recombinase activity was found in CEM or CEM/DOX cells when the pJH201 transfection assay was used. The results indicate that CEM/DOX cells failed to generate V(D)J recombination although RAG1 gene is overexpressed. The mechanism of the RAG1 gene activation was not gene amplification, and no rearrangement was detected in the RAG1 gene locus. RAG1 presents homology with the yeast gene HPR1, itself homologous to yeast topoisomerase I and responsible for the control of recombination in somatic cells. Since DNA topoisomerases are themselves involved in the control of DNA topology, recombination and DNA repair, the possible coactivation of RAG1 and topoisomerase I genes in CEM/DOX cells is discussed.

Influence of positive stress on nucleosome assembly.

Nucleosome assembly on closed circular DNA has been studied using reconstitution protocols which involve salt gradient dialysis in the presence or absence of urea. The closed circular DNA contained either +3 or -14 supercoils. The nucleosomal content of the DNA was assayed by measuring the superhelical content in the presence of topoisomerase I as a function of time and temperature and by measuring the DNA fragment size after DNase I digestion. Positively coiled DNA appears to contain an altered "open" nucleosome structure as compared to the negatively coiled DNA. Formaldehyde fixation of the reconstituted complexes was also done in an attempt to freeze the nucleosomal state prior to topoisomerase I or DNase I addition. The data from these additional experiments are consistent with a conclusion that an altered nucleosomal structure exists on positively supercoiled DNA. Control experiments are included to verify the efficiency of formaldehyde fixation in preserving the structure in the reconstituted complexes. The physiological relevance of positive stress and its effect on nucleosome structure are discussed.

Purification and characterization of the HU-like protein HPB9 from the Bacillus subtilis nucleoid

The Bacillus subtilis HPB9 is the major heat-stable and acid-soluble protein associated with the nucleoid isolated at low ionic strength. The abundance of the protein in the cell is estimated to about 20000 monomers per cell (Salti et al. (1985) J. Gen. Microbiol. 131, 581–590). The protein cross reacts specifically with the antiserum against the Bacillus globigii HBg. Moreover, HPB9 is able to introduce negative supercoiling in a relaxed covalently closed circular DNA, in the presence of topoisomerase I as demonstrated by one and two-dimensional electrophoresis. These results indicate that the nucleoid associated protein HPB9 is an HU-like protein and could be involved in the DNA compaction.

Identification and purification of DBF-A, a double-stranded DNA-binding protein from Saccharomyces cerevisiae.

Using oligonucleotide affinity chromatography with DNase I footprinting as an assay we have looked for proteins that interact with sequence elements within the yeast origin of replication, autonomously replicating sequence 1 (ARS1). In this work we describe a protein that binds with high affinity to DNA but displays only moderate sequence specificity. It is eluted at 0.7 M salt from an ARS1 oligonucleotide column. Footprinting analysis on ARS1 at a high protein concentration revealed at least three sites of protection flanking element A and its repeats. Element A itself is rendered hypersensitive to DNase I digestion upon protein binding. This pattern is also observed for the H4 and HMR-E ARSs, suggesting that the protein alters the DNA conformation at element A and its repeats. The affinity-purified fraction is also capable of supercoiling a relaxed, covalently closed plasmid in the presence of topoisomerase. Highly purified preparations of the protein are enriched in an 18-kDa polypeptide which can be renatured from a denaturing gel and shown to bind ARS1 DNA. We have designated this protein DBF-A, DNA-binding factor A.

Expression of topoisomerase II, catalase, metallothionein and thymidylate-synthase in human squamous cell lung carcinomas and their correlation with doxorubicin resistance and with patients' smoking habits.

Forty-eight human squamous cell lung carcinomas of previously untreated patients were analyzed for resistance to doxorubicin and for the presence of topoisomerase II (Topo II), metallothionein (MT), thymidylate synthase (TS) and catalase (Cat). Significant correlations exist between resistance to doxorubicin measured by the in vitro short-term test and overexpression of MT and TS measured by immunohistochemistry. No significant correlation was found between resistance and expression of Topo II or Cat. No significant interrelationship between smoking habits of patients and expression of Topo II, MT, TS or Cat was found.

Transcription-induced conformational change in a topologically closed DNA domain.

We have tested in vitro the occurrence of a B-to-Z transition in a region of alternating purines and pyrimidines as a consequence of transcription-induced negative supercoiling. By using a monoclonal antibody as a specific Z-DNA stabilizing agent, we demonstrate that the formation of left-handed DNA can transiently occur when a topologically unconstrained template is transcribed. The B-to-Z transition, observed in a subpopulation of templates, appears to be induced by negative supercoiling generated in the wake of an elongating T7 RNA polymerase. Consistent with this, the presence of topoisomerases during the transcription period prevents the change in DNA conformation. These data agree with the 'twin-supercoiled-domain' model for transcription of Liu and Wang (1). Interestingly, our results suggest that the diffusion rate of transcription-induced superhelical twists must be relatively slow compared to their generation, and that under in vitro conditions localized transient supercoiling can reach unexpectedly high levels.

HU and IHF: similarities and differences. In Escherichia coli, the lack of HU is not compensated for by IHF

HU is one of the most abundant DNA binding proteins in Escherichia coli. Like the histones, HU is able to condense DNA in vitro and to introduce negative supercoiling in covalently closed circular, relaxed DNA molecules in the presence of topoisomerase I. HU is well conserved in all prokaryotes but surprisingly, it is also homologous to another E. coli DNA-binding protein, IHF. Contrary to HU, IHF shows sequence specificity and is much less abundant that HU. Both are heterodimers and all four polypeptide chains probably arose from a common ancestor. The question we raised was whether IHF could supply the main functions of HU in its absence. The answer seems to be negative for the following reasons. We did not observe any significant regulation of expression of the genes coding for HU by IHF, or vice-versa. The structures that these two proteins form with double-stranded or single-stranded DNA are completely different. Finally, overexpression of IHF does not relieve the growth defects observed in HU-less mutants. However, it can be speculated from our results that even if the two proteins are not equivalent and cannot replace one another, both could stimulate (or inhibit) some specific protein-DNA interactions or affect the DNA-binding properties of one another.

Binding of DNA in vitro by a small, acid-soluble spore protein from Bacillus subtilis and the effect of this binding on DNA topology

The DNA within spores of Bacillus subtilis is complexed with a large amount of alpha/beta-type small, acid-soluble spore protein (SASP). Measurement of the interaction of a purified alpha/beta-type SASP with DNA in vitro by a filter binding assay showed that the binding saturated at one molecule of SASP per approximately 5 bp. SASP-DNA binding did not require a divalent cation, was optimal at pH 6.7, and was unaffected by salt up to 400 mM. Binding of SASP to relaxed plasmid DNA in the presence of topoisomerase I resulted in the introduction of 18 (for plasmid pUC19) or 36 (for plasmid pUB110) negative supertwists, a superhelical density similar to that found in several plasmids isolated from spores. The SASP-dependent introduction of negative supertwists did not require a divalent cation, was unaffected by salt, and also gave a value of one molecule of SASP per approximately 5 bp at saturation. There was at least one slow step in the binding of SASP to DNA as seen in both the filter binding and supercoiling assays.

Simian Virus 40 Origin Auxiliary Sequences Weakly Facilitate T-Antigen Binding but Strongly Facilitate DNA Unwinding

The complete simian virus 40 (SV40) origin of DNA replication (ori) consists of a required core sequence flanked by two auxiliary sequences that together increase the rate of DNA replication in monkey cells about 25-fold. Using an extract of SV40-infected monkey cells that reproduced the effects of ori-auxiliary sequences on DNA replication, we examined the ability of ori-auxiliary sequences to facilitate binding of replication factors and to promote DNA unwinding. Although the replicationally active form of T antigen in these extracts had a strong affinity for ori-core, it had only a weak but specific affinity for ori-auxiliary sequences. Deletion of ori-auxiliary sequences reduced the affinity of ori-core for active T antigen by only 1.6-fold, consistent with the fact that saturating concentrations of T antigen in the cell extract did not reduce the stimulatory role of ori-auxiliary sequences in replication. In contrast, deletion of ori-auxiliary sequences reduced the efficiency of ori-specific, T-antigen-dependent DNA unwinding in cell extracts at least 15-fold. With only purified T antigen in the presence of topoisomerase I to unwind purified DNA, ori-auxiliary sequences strongly facilitated T-antigen-dependent DNA conformational changes consistent with melting the first 50 base pairs. Under these conditions, ori-auxiliary sequences had little effect on the binding of T antigen to DNA. Therefore, a primary role of ori-auxiliary sequences in DNA replication is to facilitate T-antigen-dependent DNA unwinding after the T-antigen preinitiation complex is bound to ori-core.